Basic Research—Biology The Expression of Macrophage and Neutrophil Elastases in Rat Periradicular Lesions Taisuke Morimoto, DDS, PhD,* Masahiro Yamasaki, DDS, PhD,* Kazuhiko Nakata, DDS, PhD,*,† Masahito Tsuji, DDS, PhD,* and Hiroshi Nakamura, DDS, PhD*,† Abstract Macrophage elastase and neutrophil elastase are periradicularin- lesion evoked by bacterial infection consists of periapical inflamma- volved in tissue destruction in periradicular lesions.A tionThe with destruction of the periodontal ligament and resorption of the alveolar purpose of this study was to examine these boneelastases and root1). (The periodontal ligament is a dense connective tissue localized immunohistochemically during development of perira-between the cementum and the alveolar bone and supports the tooth. The destruction dicular lesions induced in rat mandibular first ofmolar the byapical periodontal ligament is initiated by degradation of the extracellular matrix pulpal exposure for 7, 14, 21, 28, and 42 (ECM).days. Histo-E C composedM i s mainly of collagen fibers, and elastic-system fibers in- logically, periapical inflammation developed from cluding7 to oxytalan fibers and elastin-containing fibers (ie, elastic and elaunin fibers) 21 days and then subsided after 28 days. Theare areaubiquitous of components of this 2ligament ). Elastic-system ( fibers have been these lesions gradually increased from 7 to 28 investigateddays and in the rat molar periodontal ligament including its3 ). apical portion ( subsequently decreased at 42 days. Macrophage Theelas- enzymes of ECM degradation are both serine proteinases and matrix metallo- tase was first detected at 7 days and predominantlyproteinases (MMPs). shown from 14 to 28 days, whereas neutrophil elastaseMMPs are a family of zinc-dependent endopeptidases that possess the ability to gradually increased from 14 to 28 days. Macrophage degrade all components of the ECMs and play important roles in many physiologic and elastase was significantly greater than neutrophil elas- pathological processes4, ().5 Especially, MMP-2 (geratinase A), -7 (matrilysin), -9 tase from 7 to 21 days. These results suggest that (geratinase B), and -12 (macrophage metalloelastase) have elastinolytic properties macrophage elastase was enhanced from an early stage during the development of these lesions (and6). thatMMP-12 was discovered in thioglycolate-stimulated mouse peritoneal macro- neutrophil elastase was related to the expansionphages of7(). Murine MMP-12 complementary DNA was8), clonedand, subsequently,( periapical tissue destruction including bone resorption.human and rat homologues of MMP-12 complementary DNA 9 were–11). reported ( (J Endod 2008;34:1072–1076) MMP-12 has the ability to degrade ECM components by12 )attacking and otherelastin ( basement membrane components such as fibronectin or collagen type V but not gelatins Key Words (13, 14). Collagen type IV or laminin is also degraded by murine or human MMP-12 Elastase, macrophage, neutrophil, periradicular lesion,(13, 14) but not by rat 11MMP-12). Macrophages ( with those proteolytic activities are rat able to penetrate basement membranes and invade 14several). Varioustissues roles( of MMP-12 in disease models have been revealed to date, such as the development of lung emphysema15 ),( prevention of tumor growth through angiostatin generation (16, 17), and degeneration of the aortic medium in abdominal18 ).aortic aneurysm ( From the *Department of Endodontics and †Research In- By using the complementary DNA microarray analysis, MMP-12 in rat periradicular stitute of Advanced Oral Science, Aichi-Gakuin University lesion was upregulated compared with healthy19 ). controlBut, to( date, few reports School of Dentistry, Nagoya, Japan. Supported by “AGU High-Tech Research Center” Project have been published with respect to the role of MMP-12 in periodontal and periradicu- for Private University: matching fund subsidy from MEXT (Min- lar tissues. istry of Education, Culture, Sports, Science and Technology), Neutrophil elastase, which is involved in the degradation of elastin, is one of the 2003–2007. Address requests for reprints to Dr. Hiroshi Nakamura, serine proteinases and it has strongly degradative action toward several proteins. Se- Department of Endodontics, School of Dentistry, Aichi-Gakuin creted cathepsin G and proteinase 3 also have an elastinolytic property in serine pro- University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651 teinase 20(). Polymorphonuclear leukocytes (or neutrophils) contain an abundance of Japan. E-mail address: [email protected]. lysosomal granules filled with proteolytic enzymes and other components. Neutrophil 0099-2399/$0 - see front matter Copyright © 2008 American Association of Endodontists. granules can be differentiated into specific and azurophilic (or primary) granules, the doi:10.1016/j.joen.2008.06.012 latter containing the serine proteinases such as elastase and cathepsin G as well as other enzymes 21(). Neutrophil elastase secreted by activated neutrophils can bind to and degrade ECM. This protease has wide substrate specificity and has strong competency in degrading a variety of the proteins including22). Thereelastin is( a possibility that it evokes the severe tissue destruction seen 23in) pulpitisand periradicular ( lesions (24). In human periapical disease, neutrophil elastase levels of periapical exudates associated with clinical symptoms25) and( there may be a possibility that it serves as a diagnostic marker (26). The purpose of the present study was to examine the involvement of macrophage elastase and neutrophil elastase in the development of periradicular lesions in rats. 1072 Morimoto et al. JOE — Volume 34, Number 9, September 2008 Basic Research—Biology Material and Methods primary polyclonal antibodies reactive with macrophage elastase The Induction of Periradicular Lesions (sc-8839) and neutrophil elastase (sc-9521) were obtained from Santa Twenty-four male Wistar rats, weighing about 250 g, were used in Cruz Biotechnologies (Santa Cruz, CA). Normal goat serum was used as the present study. Periradicular lesions were induced as described pre- the negative control. The sections were stained by use of ABC Staining viously (27, 28). In brief, all animals were anesthetized, and the pulpal System (sc-2018, Santa Cruz Biotechnologies). The slides were exposed tissues in the left and right mandibular first molars were exposed so that to hydrogen peroxide in deionized phosphate-buffered saline (PBS) for perforation of the furcation would be avoided. The exposed areas were 10 minutes to quench the endogenous peroxidase activity and subse- left open to the oral environment until the animals were killed. quently reacted with 1.5% goat serum diluted in PBS for an hour to block nonspecific antibody binding. These slides were incubated with Tissue Preparation and Immunohistochemistry each primary antibody for 12 hours, followed by biotinylated antibody Animals were sacrificed at 7, 14, 21, 28, and 42 days (each period, for 30 minutes. Then, they were incubated with avidin-biotinylated per- n ϭ 4) after the pulpal exposure. Animals without the exposure were oxidase complex for 30 minutes. For the final chromogenic reaction, used as the control (day 0, n ϭ 4). At the time of sacrifice, the mandi- the slides were exposed to the substrate solution consisting of diamino- bles of all animals were removed and fixed in periodate-lysine-parafor- benzidine-tetrahydrochloride and hydrogen peroxide and were coun- maldehyde solution for 24 hours at 4°C. The mandibles were decalci- terstained with hematoxylin. The periapical tissue of the mesial root of fied by being soaked in 5% EDTA solution until complete decalcification the mandibular first molar was examined for both antigen-positive cells. (approximately 60 days) at 4°C. Then, these were embedded in OCT compound (Miles Scientific, Naperville, IL) and sectioned serially at 5 Histometry ␮m in the mesiodistal plane. Quantitative analysis was performed on 4 serial sections from each The presence of macrophage elastase and neutrophil elastase molar. The area of the periradicular lesion was measured histometri- in the periapical tissue were examined immunohistochemically. The cally as described previously (26, 27). The area of the lesion was the Figure 1. Immunohistochemical staining for macrophage elastase. (A) In the mandibular first molar at 7 days after the exposure, some elastase-expressing cells detect in the residual pulpal tissue and around the root apex. B is a high-magnification view of cropped area in A. These cells gradually increase at (C) 14 and (D) 21 days. (E) By 28 days, these cells increase in number and are observed in and around the abscess. F is a high-magnification view of cropped area in E. G is a cropped area in F. The arrows indicate macrophage elastase-positive cells. These cells are round-shaped monocytes in high magnification. By 42 days, these cells decrease in number, but some still remain in the abscess (H). A, C, D, E, and H scale bars ϭ 1.0 mm. B and F scale bars ϭ 0.25 mm. G scale bar ϭ 0.025 mm. JOE — Volume 34, Number 9, September 2008 Expression of Elastases in Rat Periradicular Lesions 1073 Basic Research—Biology periodontal ligament between the root apex and alveolar bone in the flammation and alveolar bone resorption were observed. By 21 days, a mesial root. The cells reactive with antimacrophage and antineutrophil small abscess was found around the root apex. Severe inflammation and elastases were counted in an area 0.6 mm square